Similar interaction chromatography of proteins: A cross interaction chromatographic approach to estimate the osmotic second virial coefficient

• Self-interaction chromatography and cross-interaction chromatography were used to determine osmotic second virial coefficient (B22, B23, B32, B33) values for 4 protein systems.
• B22 values can be reliably estimated from B23 and B32 values determined by cross-interaction chromatography.
• Estimated B22 data obtained were accurate enough to allow for a first order classification of protein solution stability.

Self-interaction chromatography (SIC) has established itself as an important experimental technique for the measurement of the second osmotic virial coefficients B22. B22 data are critical for understanding a range of protein solution phenomena, particularly aggregation and crystallisation. A key limitation to the more extensive use of SIC is the need to develop a method for immobilising each specific protein of interest onto a chromatographic support. This requirement is both a time and protein consuming constraint, which means that SIC cannot be used as a high throughput method for screening a wide range of proteins and their variants. Here an experimental framework is presented for estimating B22 values using Similar Interaction Chromatography (SimIC). This work uses experimental B23 and B32 data for lysozyme, lactoferrin, catalase and concanavalin A to reliably estimate B22 using arithmetic mean field approximations and is demonstrated to give good agreement with SIC measurements of B22 for the same proteins. SimIC could form the basis of a rapid protein variant screening methods to assess the developability of protein therapeutic candidates for industrial and academic researchers with respect to aggregation behaviour by eluting target proteins through a series of well-characterised protein immobilized reference columns.